Project 1: Development of Nucleoside-Modified mRNAs Encoding Sequential HIV-1 Envelopes for Initiation of V3-glycan Neutralizing Antibody Lineages A major goal of HIV-1 vaccine development is to induce broadly neutralizing antibodies (bnAbs). This IPCAVD team has immunogens that can trigger unmutated common ancestors (UCAs) of bnAb B cell lineages, a key approach for an HIV vaccine. One such sequential Env immunogen termed, EnvSeq-3, is derived from the study of the CH848 HIV-1-infected individual who made the DH270 HIV V3-glycan-targeted bnAb. We hypothesize that use of priming immunogens that bind to the UCA of the DH270 B cell lineage followed by sequential modified mRNA gp160 or gp140 trimer-based immunogens will result in the initiation of V3-glycan bnAb B cell lineages in humans. ? Specific Aim 1. Design and formulate sequential nucleoside-modified mRNAs encoding two gp160s or soluble chimeric, stabilized trimers that bind to intermediate antibodies of the DH270 B cell lineage. Nucleoside-modified mRNA encoding the first 2 trimeric lineage immunogens after the V3-glycopeptide will be optimized for high level expression using established techniques. ? Specific Aim 2. Determine if synthetic UCA-binding glycopeptides combined with GLA/SE adjuvant will prime DH270 UCA B cells for boosting with Env trimer-encoding mRNAs in DH270 UCA VH and VL knock-in mice. Glycopeptide and mRNA immunogens will be analyzed including combinations to confirm in DH270 UCA VH + VL knock-in (KI) mice their induction of Tfh cells and selection of antibodies that target the V3-glycan bnAb site. The two selected mRNAs will be produced CGMP in Project 2. ? Specific Aim 3. Test sequential immunogens in outbred rhesus macaques to optimize immunization schemes for use in a phase I clinical trial in year 5. We believe that modified mRNA-LNPs encoding V3-glycan lineage immunogens will generate potent CD4+ Tfh and antibody responses, and will initiate V3-glycan bnAb lineages in bnAb KI mice and in humans. This project will use a promising sequential immunogen design in the form of nucleoside-modified mRNAs to overcome barriers to bnAb development. If successful, the use of nucleoside-modified mRNAs will transform HIV-1 vaccine development.